Abstract Despite the extraordinary conservation of sleep across evolution and the established importance of sleep to human health, the needs fulfilled by sleep remain one of the biggest mysteries in neuroscience. In fact, no consensus has emerged about either the neuroanatomical origins or the molecular basis by which sleep need is sensed and discharged. It is a vexing but understandable problem. Screens for candidate genes are too time-consuming and expensive to be practical in vertebrates. And among cheaper, more genetically tractable model organisms, most assays are designed only to identify mutations that constitutively disturb daily sleep, not genes that regulate the mysterious homeostatic process that senses and responds to sleep need. To address this major deficiency, my lab has developed a simple, robust, high-throughput thermogenetic assay for measuring sleep need in Drosophila. Using this assay we have demonstrated that arousal-promoting neurons surprisingly only rarely drive sleep homeostasis. In this proposal we identify these rare neurons as cells that express the gene ppk, describe their likely sensory role, and highlight experiments to determine the types of information that these neurons transduce to drive sleep need. Furthermore, by suppressing the activity of various neurons in the brain, we have also identified postsynaptic effectors of ppk neurons. Using a genetically encoded Ca2+ sensor we will confirm the functional connectivity between these different cellular regulators of sleep need. Using a combination of forward genetic screening and mass spectrometry, we have also identified molecules that appear to be required to mediate sleep homeostasis. We will confirm the functions of these molecules in regulating sleep need and identify the subset of proteomic changes that occur during sleep homeostasis due to these functions. Lastly, we have shown that sleep homeostasis is required following sleep deprivation in order for subsequent memory formation to occur. Thus, we will determine whether mechanisms underlying the two processes are likely to be shared. Specifically, we will reduce the activity of newly discovered neurons and molecules we have implicated in sleep homeostasis to determine if they are also required for associative memory formation. Defining mechanisms underlying sleep homeostasis and their relation to cognition, as proposed in this grant, would represent major breakthroughs in neuroscience and may facilitate the development of novel pharmacotherapies to intervene in sleep-related disorders.